MAS is classified into 3 types based on disease prevalence [3]. Type 1 MAS includes conditions like myasthenia gravis, thymoma, polymyositis, and giant-cell myocarditis. Type 2 MAS encompasses Sjögren's syndrome, rheumatoid arthritis, primary biliary cirrhosis, scleroderma, and autoimmune thyroid diseases. Type 3 MAS includes autoimmune thyroid disease, myasthenia gravis and/or thymoma, Sjögren's syndrome, pernicious anemia, immune-mediated thrombocytopenia (ITP), Addison’s disease, type 1 diabetes mellitus, vitiligo, immune-mediated hemolytic anemia (IMHA), systemic lupus erythematosus (SLE), and dermatitis herpetiformis. Understanding these categories helps clinicians predict and detect new autoimmune diseases in patients with 2 existing autoimmune conditions.

In veterinary medicine, MAS reports are scarce, and the management of MAS in dogs often relies on human criteria [4,5]. The heritability of autoimmune diseases in purebred dogs is around 50% in studied breeds, with environmental and epigenetic factors also significant [5]. Here, we present a case involving a dog suspected of having type 3 MAS, featuring IMHA, ITP, and immune-mediated neutropenia (IMN) alongside a history of inflammatory bowel disease (IBD).

A 6-year-old neutered male, Shih-Tzu, presented to our clinic with a 10-day history of melena, petechiae, and ecchymosis. Diagnosed with IBD 4 years prior, the dog had chronic gastrointestinal symptoms unresponsive to a hypoallergenic diet trial, confirmed by histopathology. Managed with an anallergenic diet, the dog was not on medication for IBD at presentation. Physical examination revealed petechiae and ecchymosis, though capillary refill time, mucous membrane color, and blood pressure were normal. Hematuria was noted once.

Laboratory analyses were conducted to determine the cause of the coagulopathy. A complete blood count revealed severe thrombocytopenia (1 × 10³/μL [reference interval, RR; 148-484 × 10³/μL]), lymphocytosis, monocytosis, and reticulocytosis. Hematocrit was within normal limits, and the peripheral blood film showed no platelet clumping. The coagulation profile indicated a delayed activated partial thromboplastin time (95 [RR, 60-93] seconds), though prothrombin time was normal. Serum biochemical values were mostly normal, except for elevated gamma-glutamyl transferase. Tests for tick/vector-borne diseases were negative. After excluding other potential causes of severe thrombocytopenia, a tentative diagnosis of ITP was made. Treatment with prednisolone and mycophenolate mofetil (MMF), along with vincristine, pantoprazole, and maropitant for gastrointestinal protection, was initiated. Blood platelet levels began to rise 3 days after treatment initiation and normalized within 6 days. The dog was managed without recurrence.

The dog returned 4 years later when the dog was 10 years old with lethargy and diarrhea. Physical examination was largely unremarkable except for mild tachypnea. Laboratory analyses showed microcytic hypochromic regenerative anemia with reticulocytosis (manual packed cell volume 24%, reticulocytes 210.7 × 10³/μL [RR, 10-110 × 10³/μL]), leukocytosis with neutrophilia, and a left shift in differential count. Prominent spherocytosis and polychromasia were noted in red blood cell morphology (Fig. 1). Serum biochemical analyses revealed elevated alkaline phosphatase and hypertriglyceridemia. IMHA was provisionally diagnosed after ruling out other causes of spherocytosis. Treatment with prednisolone and MMF was initiated, leading to improvement. The dog was subsequently managed well without recurrence, and medication was tapered and discontinued after 6 months.

The dog returned 2 years later, at the age of 12, with fever and neutropenia. Anorexia persisted for 3 days. Examination revealed fever (rectal temperature 39.6°C), tachypnea, and hypertension, with mild dehydration suggested by delayed skin turgor. Laboratory tests showed leukopenia (1.94 × 10³/μL [RR, 5.05-16.76 × 103/μL]) with neutropenia (0.48 × 10³/μL [RR, 2.95-11.64 × 10³/μL]) and lymphopenia (0.80 × 103/μL [RR, 1.05-5.10 × 10³/μL]). Serum biochemistry indicated increased C-reactive protein (CRP) and elevated alkaline phosphatase (Table 1). To diagnose the cause of neutropenia, it was considered infectious diseases, neoplasia, drug or toxin exposure, severe inflammation, bone marrow disorders, and IMN. Comprehensive tests were conducted, including checks for pancreatitis, urinalysis, urine culture, and vector-borne diseases, all of which returned negative results. Blood cultures and a tick/vector polymerase chain reaction test showed no growth or infection. Tests for antinuclear antibodies and rheumatoid factor were also negative, and diagnostic imaging was unremarkable. Despite these findings, infection remained a possibility, so amoxicillin-clavulanate (12.5 mg/kg intramuscular every 12 hours) and doxycycline (5 mg/kg per oral every 12 hours) were injected. Over the next 2 days, there was no improvement despite additional antibiotics at the same dose and frequency. In light of the absence of notable improvement after the introduction of antibiotics, a determination was made to administer prednisolone (2.0 mg/kg subcutaneously once daily) every visit, with due consideration for the potential existence of an immune-mediated disease. Following the initiation of prednisolone, there was an amelioration in the temperature and neutrophil counts, accompanied by a decrease in CRP levels. After 12 days, CRP levels had returned to normal range, prednisolone was reduced (1.75 mg/kg per oral every 24 hours) and antibiotics were discontinued. Since then, the medication was rapidly tapered due to suspected symptoms of pancreatitis and discontinued on day 59 and the disease has been well controlled without recurrence (Fig. 2). Sepsis, tick-borne diseases, pancreatitis, and SLE were ruled out as causes of neutropenia. Malignancy, drugs, and toxins were unlikely. Myelodysplasia was improbable due to neutrophil count improvement. With a positive response to corticosteroids, a provisional IMN diagnosis was made. The dog is responding well to treatment and remains healthy.

This case highlights a dog with a history of IBD that sequentially developed ITP, IMHA, and IMN, suggesting a diagnosis of type 3 MAS. In humans, IBD is often associated with autoimmune diseases, and ulcerative colitis frequently co-occurs with type 3 MAS [1,3]. In veterinary medicine, the understanding of MAS in dogs is limited, leading to reliance on human classification and treatment protocols [4]. Based on human criteria, the dog in this case was suspected of having type 3 MAS based on the diagnosis of 3 autoimmune diseases (ITP, IMHA, and IMN) and a history of IBD.

Autoimmune cytopenia arises from autoantibodies targeting blood cells, necessitating the exclusion of other conditions for diagnosis [6]. The presence of other autoimmune diseases can aid in diagnosing autoimmune cytopenia. Approximately 25% of humans and dogs with idiopathic IMN develop thrombocytopenia [7]. IMHA and ITP, known as Evans syndrome, frequently co-occur in veterinary medicine [7,8]. In a study of 5 dogs with IMN, all had other immune-mediated diseases [9], and another study suggested that 30% of dogs with ITP have co-occurring IMHA [10].

In this case, the dog also had a history of IBD before the onset of autoimmune cytopenia. In human patients with ulcerative colitis, the prevalence of autoimmune cytopenia is reportedly 28% [11]. According to one report, the prevalence of IMHA in patients with ulcerative colitis is 150 per 100,000 patients, which is higher than the general prevalence of IMHA (17 per 100,000) [12]. In one veterinary study, the presence of anti-red blood cell (RBC) antibodies was confirmed in 76% of dogs with IBD [11]. However, the association between autoimmune cytopaenia and IBD remains unclear, although the cross-reactivity between the intestinal microbiome and the E antigen on the surface of RBCs may partially explain the association between intestinal and IMHA activity [12]. According to another theory, chronic inflammation due to intestinal disease may induce changes in the erythrocyte membrane due to oxidative stress, leading to the production of anti-erythrocyte antibodies [11].

Genetic factors are also implicated in MAS development. Studies in humans have identified clusters of polyautoimmunity and genetic loci associated with autoimmunity [13]. In dogs, certain breeds are more prone to immune disorders, and the dog leukocyte antigen (DLA) complex is linked to autoimmune diseases [5]. Specific alleles, such as DLA-79*001:02, associate with multiple immune-mediated diseases [14]. However, the genetic basis of the presented case is unclear due to the lack of family history and genetic analysis. This study had limitations, including unclear diagnostic criteria for IMHA and the lack of bone marrow evaluation. Despite these, the dog responded well to treatment, supporting the diagnosis.

This study had limitations. The diagnostic criteria for IMHA were unclear, and hemolysis evidence was lacking, but regenerative anemia with spherocytosis led to an IMHA diagnosis due to the response to immunosuppressants. Bone marrow wasn’t evaluated, but a blood smear showed normal morphology, and corticosteroid response suggested low abnormality risk. The MMF dosage was higher than typical (7-9 mg/kg twice daily), but the dog was carefully monitored for side effects.

In conclusion, this case highlights the importance of being aware of and monitoring autoimmune diseases in dogs with a history of immune-mediated conditions. It also highlights the potential for using human MAS criteria to guide veterinary diagnosis and treatment, suggesting areas for future research in genetic and environmental influences on autoimmune disease in dogs.